miR-92对神经母细胞瘤细胞增殖作用的研究

Effect of miR-92 on the proliferation and apoptosis of human neuroblastoma cells

目的探讨microRNA-92（miR-92）对神经母细胞瘤细胞增殖的促进作用，并预测miR-92的靶基因。方法人工设计合成miR-92的反义寡核苷酸（anti—miR-92寡核苷酸），利用脂质体LipofectamineTM2000转染神经母细胞瘤SK-N-SH和SK-N-BE2细胞系，利用实时定量PCR检测转染后细胞内miR-92的表达水平，并通过噻唑蓝（MTT）比色法和流式细胞仪检测细胞的增殖和凋亡情况；利用生物信息学方法对miR-92的候选靶基因进行预测。结果与对照组细胞相比，两种神经母细胞瘤细胞转染anti-miR-92后，miR-92的水平降低约70％，MTT检测细胞的活性降低约20％，流式分析仪检测发现增殖的细胞数降低约50％～60％，而细胞凋亡数增加约2～3倍，差异均具有统计学意义（P〈0．05）。通过生物信息学和对靶基因潜在功能分析，初步选定BCL2-like11（BCL2L11）和phosphatase and tensin homolog deleted on chromosome ten （PTEN）作为是miR-92的候选靶基因。结论抑制miR_92的表达导致神经母细胞瘤细胞的增殖降低，凋亡增加，从而可能进一步影响神经母细胞瘤的生长，miR-92可能具有作为临床上神经母细胞瘤治疗分子标记物的潜在功能，为以后更有意义的分子治疗奠定基础。

Objective To investigate the effects of miR-92 on the proliferation and apoptosis in human neuroblastoma （NB） cells, and to predict its putative targets. Methods Antisense oligonucle- otides of miR-92 were synthesized and transfected into SK-N-SH and SK-N-BE2 cells, then real time PCR was performed for the detection of miR-92 expression. Cell proliferation and apoptosis were de- termined using MTT assay and flow cytometry. The putative targets for miR-92 were predicted using bioinformatics. Results Cell viability and proliferation of anti-miR-92 transfected cells were reduced by 20% and 50%-60%, respectively, while apoptosis increased by 2-3 fold （P〈0. 05）. Using bioinfor- matics, BCL2-1ike 11 （apoptosis facilitator） （BCL2L11） and PTEN were selected as the candidate tar- gets for miR-92. Conclusions Our data indicate that the inhibition of miR-92 can suppress NB cell proliferation and increase cell apoptosis, suggesting miR-92 may play important roles in NB growth. In addition, miR-92 may serve as a novel biomarker for therapeutic strategy of NB patients.